Mechanisms of PTHrP-induced inhibition of smooth muscle contractility in the guinea pig gastric antrum.

BACKGROUND: Parathyroid hormone-related protein (PTHrP) that causes hypercalcemia of malignancy appears to function as an endogenous smooth muscle relaxant. For example, PTHrP released upon bladder wall distension relaxes detrusor smooth muscle to accommodate urine. Here, we explored mechanisms underlying PTHrP-induced suppression of the smooth muscle contractility in the gastric antrum that also undergoes a passive distension. METHODS: Effects of PTHrP on phasic contractions and electrical slow waves in the antral smooth muscle of the guinea pig stomach were studied using isometric tension and intracellular microelectrode recordings, respectively. Fluorescent immunohistochemistry was also carried out to identify the distribution of PTH/PTHrP receptors. KEY RESULTS: Parathyroid hormone-related protein (1-100 nM) reduced the amplitude of phasic contractions and the basal tension. Nω -nitro-l-arginine (L-NA, 100 µM), a nitric oxide (NO) synthase inhibitor, or 1H-[1,2,4]oxadiazolo-[4, 3-a]quinoxalin-1-one (ODQ, 10 µM), a guanylate cyclase inhibitor, diminished the PTHrP (10 nM)-induced reduction in the amplitude of phasic contractions. SQ22536 (300 µM), an adenylate cyclase inhibitor, attenuated the PTHrP-induced reduction in basal tension. The combination of ODQ (10 µM) and SQ22536 (300 µM) inhibited the PTHrP-induced reductions in both phasic contractions and basal tension. PTHrP (100 nM) had no inhibitory effect on the electrical slow waves in the antral smooth muscle. PTH/PTHrP receptors were expressed in cell bodies of PGP9.5-positive neurons in the myenteric plexus. CONCLUSIONS & INFERENCES: Parathyroid hormone-related protein exerts its inhibitory actions on the antral smooth muscle via both nitric oxide-cyclic guanosine monophosphate (NO-cGMP) and cyclic adenosine monophosphate (AMP) pathways. Thus, PTHrP may act as an endogenous relaxant of the gastric antrum employing the two complementary signaling pathways to ensure the adaptive relaxation of stomach.

Autonomic and sensory nerve modulation of peristalsis in the upper urinary tract.

The primary function of the upper urinary tract is to propel urine and various water-soluble toxic compounds from the kidneys to the bladder for storage and evacuation to maintain body ionic balance and contribute to the regulation of blood volume and pressure. The mechanism by which the upper urinary tract propels urine has long been considered to be myogenic in origin as peristaltic contractions in vivo and in vitro (pyeloureteric peristalsis) propagate in a manner little affected by drugs that block nerve conduction or the sympathetic and parasympathetic transmission. However, it is now well established that the release of intrinsic prostaglandins and neuropeptides from primary sensory nerves (PSNs) helps to maintain pyeloureteric peristalsis. Electrical field stimulation of PSNs evokes species-specific positive inotropic and chronotropic effects that have been attributed to release of excitatory tachykinins superimposed on negative inotropic and chronotropic effects associated with the release of calcitonin gene related peptide (CGRP), a rise in cellular cyclic-adenosine monophosphate (cAMP) and a protein kinase A-dependent activation of glibenclamide-sensitive ATP-dependent K+ (KATP) channels. This review summarises the existing evidence of the nervous control of the upper urinary tract and recent evidence suggesting that the autonomic innervation may indirectly modulate pyeloureteric peristalsis via the activation of PSN nicotinic receptors and via the modulation of KV7 channels located on interstitial cells within the renal pelvis wall.

Calcium responses in subserosal interstitial cells of the guinea-pig proximal colon.

BACKGROUND: In the subserosal layer between the longitudinal muscle layer and mesothelium, heterogeneous populations of interstitial cells are distributed. As the distribution of nerve elements in this layer is sparse as compared with the nerve plexus layer or tunica muscularis, there may be unique communication among subserosal interstitial cells (SSICs). This study aimed to explore functional properties of SSICs. METHODS: In subserosal preparations of the guinea-pig proximal colon, changes in intracellular Ca(2+) ([Ca(2+) ]i ) were visualized using Fluo-4 Ca(2+) imaging. Immunohistochemistry was also performed to identify the SSICs exhibiting Ca(2+) transients. KEY RESULTS: A majority of SSICs responded to adenosine triphosphate (ATP, 10 µM) by increasing [Ca(2+) ]i , but remained quiescent during the application of acetylcholine (10 µM). ATP-induced Ca(2+) responses were mimicked by adenosine 5'-diphosphate (10 µM), MRS2365 (10 nM) but not α, ß-methylene ATP (10 µM) or uridine triphosphate (10 µM), and could be reproduced in Ca(2+) -free solution, suggesting that ATP acts via P2Y receptors, most likely P2Y1 subtype, but not P2X receptors. Live staining of the same preparations after Ca(2+) imaging indicated the ATP-sensitive SSICs were not positive for c-Kit antibody, a specific marker for gastrointestinal interstitial cells of Cajal (ICC). Immunohistochemistry identified vimentin (mesenchymal cell marker)+/Kit- and SK3 (fibroblast-like cell (FLC) marker)+/Kit- cells that had a similar morphology to the ATP-sensitive SSICs in Ca(2+) imaging. CONCLUSIONS & INFERENCES: A majority of the SSICs in the guinea-pig proximal colon, presumably FLC, are capable of responding to ATP and thus may contribute to smooth muscle relaxation upon stimulation with ATP released from non-neuronal cells.

Nicotinic receptor activation on primary sensory afferents modulates autorhythmicity in the mouse renal pelvis.

BACKGROUND AND PURPOSE: The modulation of the spontaneous electrical and Ca(2+) signals underlying pyeloureteric peristalsis upon nicotinic receptor activation located on primary sensory afferents (PSAs) was investigated in the mouse renal pelvis. EXPERIMENTAL APPROACH: Contractile activity was followed using video microscopy, electrical and Ca(2+) signals in typical and atypical smooth muscle cells (TSMCs and ASMCs) within the renal pelvis were recorded separately using intracellular microelectrodes and Fluo-4 Ca(2+) imaging. KEY RESULTS: Nicotine and carbachol (CCh; 1-100 µM) transiently reduced the frequency and increased the amplitude of spontaneous phasic contractions in a manner unaffected by muscarininc antagonists, 4-DAMP (1,1-dimethyl-4-diphenylacetoxypiperidinium iodide) and pirenzipine (10 nM) or L-NAME (L-Nω-nitroarginine methyl ester; 200 µM), inhibitor of NO synthesis, but blocked by the nicotinic antagonist, hexamethonium or capsaicin, depletor of PSA neuropeptides. These negative chronotropic and delayed positive inotropic effects of CCh on TSMC contractions, action potentials and Ca(2+) transients were inhibited by glibenclamide (Glib; 1 µM), blocker of ATP-dependent K (KATP) channels. Nicotinic receptor-evoked inhibition of the spontaneous Ca(2+) transients in ASMCs was prevented by capsaicin but not Glib. In contrast, the negative inotropic and chronotropic effects of the non-selective COX inhibitor indomethacin were not prevented by Glib. CONCLUSIONS AND IMPLICATIONS: The negative chronotropic effect of nicotinic receptor activation results from the release of calcitonin gene-related peptide (CGRP) from PSAs, which suppresses Ca(2+) signalling in ASMCs. PSA-released CGRP also evokes a transient hyperpolarization in TSMCs upon the opening of KATP channels, which reduces contraction propagation but promotes the recruitment of TSMC Ca(2+) channels that underlie the delayed positive inotropic effects of CCh.

Effects of imatinib mesylate on spontaneous electrical and mechanical activity in smooth muscle of the guinea-pig stomach.

BACKGROUND AND PURPOSE: Effects of imatinib mesylate, a Kit receptor tyrosine kinase inhibitor, on spontaneous activity of interstitial cells of Cajal (ICC) and smooth muscles in the stomach were investigated. EXPERIMENTAL APPROACH: Effects of imatinib on spontaneous electrical and mechanical activity were investigated by measuring changes in the membrane potential and tension recorded from smooth muscles of the guinea-pig stomach. Its effects on spontaneous changes in intracellular concentration of Ca(2+) ([Ca(2+)](i)) (Ca(2+) transients) were also examined in fura-2-loaded preparations. KEY RESULTS: Imatinib (1-10 microM) suppressed spontaneous contractions and Ca(2+) transients. Simultaneous recordings of electrical and mechanical activity demonstrated that imatinib (1 microM) reduced the amplitude of spontaneous contractions without suppressing corresponding slow waves. In the presence of nifedipine (1 microM), imatinib (10 microM) reduced the duration of slow waves and follower potentials in the antrum and accelerated their generation, but had little affect on their amplitude. In contrast, imatinib reduced the amplitude of antral slow potentials and slow waves in the corpus. CONCLUSIONS AND IMPLICATIONS: Imatinib may suppress spontaneous contractions of gastric smooth muscles by inhibiting pathways that increase [Ca(2+)](i) in smooth muscles rather than by specifically inhibiting the activity of ICC. A high concentration of imatinib (10 microM) reduced the duration of slow waves or follower potentials in the antrum, which reflect activity of ICC distributed in the myenteric layers (ICC-MY), and suppressed antral slow potentials or corporal slow waves, which reflect activity of ICC within the muscle bundles (ICC-IM), presumably by inhibiting intracellular Ca(2+) handling.

Mechanisms of excitatory transmission in circular smooth muscles of the guinea pig seminal vesicle.

PURPOSE: Cellular mechanisms of excitatory neuromuscular transmission in circular smooth muscles of the seminal vesicle were investigated. MATERIALS AND METHODS: Circular smooth muscles of the seminal vesicle of the guinea pig were isolated. Changes in membrane potential produced by transmural nerve stimulation were recorded using intracellular microelectrode techniques. Changes in the intracellular Ca ion concentration induced by transmural nerve stimulation were measured in preparations loaded with Ca indicator fura-PE3. Responses produced by bath applied norepinephrine and alpha,beta-methylene adenosine triphosphate (ATP) were also examined. RESULTS: Transmural nerve stimulation evoked excitatory junction potentials that triggered action potentials and also caused transient increases in [Ca2+] (Ca transients). Nifedipine abolished action potentials, leaving underlying excitatory junction potentials unchanged, and reduced the amplitude of Ca transients. Excitatory junction potentials were blocked by alpha,beta-methylene ATP or guanethidine but not by phentolamine. A train of transmural nerve stimulation evoked oscillatory changes in membrane potential and [Ca2+], which were abolished by phentolamine or inhibited by nifedipine. Nifedipine insensitive components were abolished by cyclopiazonic acid. Norepinephrine depolarized the membrane and elicited oscillatory potentials with an associated elevation in [Ca2+]. These responses were inhibited by nifedipine and abolished by additional application of cyclopiazonic acid. Transient depolarization with an associated increase in [Ca2+] was elicited by alpha,beta-methylene ATP and [Ca2+] responses but no potential changes were inhibited by nifedipine. CONCLUSIONS: Circular smooth muscles of the guinea pig seminal vesicle receive a projection of sympathetic nerves that release norepinephrine to initiate slow depolarization through the activation of alpha-adrenoceptors. These nerves also release ATP to elicit excitatory junction potentials. Neurally released norepinephrine and ATP are increased [Ca2+] by the influx of Ca2+ through L-type Ca2+ channels and also by the release of Ca2+ from internal stores.

Effects of isoproterenol on spontaneous excitations in detrusor smooth muscle cells of the guinea pig.

PURPOSE: Because beta-adrenoceptor agonists would be a useful tool for the pharmacological treatment of unstable bladder, we investigated the cellular mechanisms underlying beta-adrenoceptor mediated inhibition on spontaneous excitation in detrusor smooth muscle. MATERIALS AND METHODS: Detrusor smooth muscle bundles were isolated from guinea pig bladders. Changes in membrane potential were recorded using an intracellular recording technique. In preparations loaded with the calcium indicator fura-PE3 changes in the concentration of intracellular calcium ions were measured simultaneously with membrane potential. Effects of isoproterenol on spontaneous changes in the membrane potential and intracellular Ca(2+) were examined RESULTS: Detrusor smooth muscle cells exhibited spontaneous action potentials that were associated with transient increases in intracellular Ca(2+) (calcium transients). Isoproterenol, which hyperpolarized the membrane, prevented action potentials and calcium transients. This induced inhibition of calcium transients was not affected by cyclopiazonic acid. Isoproterenol induced hyperpolarization was inhibited by inhibitors of protein kinase A, N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide, hydrochloride and Rp-adenosine-3',5'-cyclic phosphorothioate. Hyperpolarization was blocked by a solution containing 30 mM. potassium but not by a range of potassium channel blockers. Ouabain and a solution of 0.5 mM. potassium also inhibited hyperpolarization. CONCLUSIONS: Our results suggest that isoproterenol prevented spontaneous action potential discharges and associated calcium transients through the activation of protein kinase A. The isoproterenol induced inhibition of intracellular Ca(2+) largely depends on the prevention of spontaneous action potentials since the contribution of the intracellular calcium store was small. Isoproterenol hyperpolarizes the membrane, probably by stimulating sodium pump activity.

Neuroeffector transmission to different layers of smooth muscle in the rat penile bulb.

1. Using intracellular recording techniques, two distinct layers of smooth muscle were identified in the rat penile bulb. The inner muscle layer (parenchyma) exhibited spontaneous action potentials, while the outer sheet (sac) was electrically quiescent. 2. In the parenchyma, transmural stimulation initiated non-adrenergic, non-cholinergic (NANC) inhibitory junction potentials (IJPs) which were abolished by Nomeganitro-L-arginine (LNA) or 1H-[1,2, 4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). The amplitude of IJPs was reduced by ouabain, dinitrophenol or decreasing the extracellular potassium concentration ([K+]o) but not by several K+ channel blockers. 3. The parenchyma also received an excitatory innervation mediated by alpha-adrenoceptors which caused a contraction that was not associated with a membrane potential change. 4. In the sac, transmural stimulation initiated two component excitatory junction potentials (EJPs) mediated by alpha-adrenoceptors and associated action potentials. The initial component was more dramatically suppressed than the secondary component by caffeine, ryanodine or cyclopiazonic acid (CPA). Lowering of the extracellular chloride concentration ([Cl-]o) selectively inhibited the rapid component of EJPs, while niflumic acid was less potent. 5. These results suggest that IJPs in the parenchyma result from the release of NO which stimulates sodium pump activity following the activation of guanylate cyclase. In the sac, the activation of alpha-adrenoceptors initiates EJPs by releasing Ca2+ from intracellular stores which activates Ca2+-activated channels.

Spontaneous and neurally activated depolarizations in smooth muscle cells of the guinea-pig urethra.

1. Membrane potential recordings were made from longitudinal smooth muscle cells of the guinea-pig urethra using conventional microelectrode techniques. 2. Smooth muscle cells of the urethra developed spontaneous transient depolarizations (STDs) and slow waves. Single unit STDs had amplitudes of approximately 5 mV and slow waves seemed to occur as amplitude multiples of single unit STDs. 3. STDs and slow waves were abolished by niflumic acid or low chloride solution and also by cyclopiazonic acid (CPA), BAPTA or high concentrations of caffeine. Lower concentrations of caffeine abolished slow waves but not STDs. Nifedipine inhibited slow waves but not STDs. 4. When stochastic properties of STDs were examined, it was found that the intervals between occurrences were not well modelled by Poisson statistics, instead the STDs appeared to be clustered. 5. Transmural stimulation evoked excitatory junctional potentials (EJPs) and triggered slow waves which were abolished by either alpha,beta-methylene-ATP or tetrodotoxin. Evoked slow waves were also abolished by caffeine, co-application of caffeine and ryanodine or by CPA which left EJPs unaffected. 6. In conclusion, smooth muscle cells of urethra exhibit STDs which are clustered rather than random events, and are the result of spontaneous Ca2+ release from intracellular stores and subsequent activation of Ca2+-activated chloride channels. STDs sum to activate L-type Ca2+ channels which contribute to the sustained phase of slow waves. Stimulation of purinoceptors by neurally released ATP initiates EJPs and also causes the release of Ca2+ from intracellular stores to evoke slow waves.

Altered electrical properties of bladder smooth muscle in streptozotocin-induced diabetic rats.

OBJECTIVE: To examine the alterations in the electrical properties of bladder smooth muscle in rats with diabetes mellitus (DM) induced by streptozotocin. MATERIALS AND METHODS: The study comprised 34 control rats and 20 with DM induced experimentally by injection with streptozotocin. At 10-15 weeks after the induction of DM, control and treated rats were killed, the bladder removed and the electrical responses of membranes from the detrusor smooth muscle recorded using intracellular microelectrodes. RESULTS: The resting membrane potential of the smooth muscle remained unaltered by DM but the frequency of spontaneous spike discharges sensitive to nicardipine was decreased. Transmural nerve stimulation elicited a purinergic excitatory junction potential (EJP) in muscles from both types of rat, but the threshold intensity of stimulation required to evoke an EJP was higher in muscle from the diabetic rat. Furthermore, EJPs large enough to trigger a nicardipine-sensitive spike potential could be elicited easily in muscles from the control rat, but not in muscles from diabetic rats. Stimulation of muscarinic receptors with exogenous acetylcholine (ACh) caused greater depolarization in muscle from diabetic rats. Stimulation of purinergic receptors with alpha, beta-methylene ATP caused a similar depolarization in both groups of rats. Application of potassium-free solution or ouabain depolarized the membrane by about 9 mV or 5 mV, respectively, in muscles from both groups of rat. Removal of potassium-free solution produced an ouabain-sensitive transient hyperpolarization which was larger in muscle from control rats, indicating that the potency of the Na-K pump was weakened in rats with DM. CONCLUSION: In the detrusor smooth muscle of rats with DM, there were fewer spontaneous spike discharges, supersensitivity of post-junctional muscarinic receptors, reduced potency of the post-junctional Na-K pump and a decrease in the release of neurotransmitter, possibly due to the impairment of prejunctional activity.

Electrical and mechanical responses produced by nerve stimulation in detrusor smooth muscle of the guinea-pig.

In smooth muscles of the guinea-pig bladder, intramural nerve stimulation generated an excitatory junctional potential (e.j.p.), action potential and twitch contraction. Nicardipine inhibited the action potential but not the e.j.p. The e.j.p. amplitude was reduced by suramin, or desensitization of the ATP receptor with receptor agonists. The amplitude of the twitch contraction was reduced by atropine, and the remainder was blocked by nicardipine. In the presence of maximally effective concentrations of atropine, the threshold concentration of acetylcholine required to produce contraction was about 10(-7) M, whereas acetylcholine concentrations greater than 10(-6) M were required to cause depolarization. It is concluded that nerve stimulation releases acetylcholine and ATP, and the former produces contraction without change in the membrane potential, while the latter generates the e.j.p. which triggers an action potential and thus elicits contractions.